Multiplex nucleic acid analysis using archived or fixed samples

Abstract

The present invention is directed to compositions and methods for multiplex analyses of nucleic acids from archival tissues.

1 - 124 . (canceled) 
     
     
         125 . A multiplex method for determining the amount of at least two target nucleic acid sequences in a sample, comprising the steps of:
 (a) providing a sample having target sequences;   (b) contacting the sample with a set of probes for each target sequence, each probe set comprising
 a first probe having a first target-specific sequence and a first universal priming sequence, and 
 a second probe having a second target-specific sequence and a second universal priming sequence, 
 to form hybridization complexes with target sequences present in the sample; 
   (c) contacting the hybridization complexes with an enzyme composition, wherein if a target sequence is present, a corresponding amplification template is formed having the first and second priming sequences;   (d) providing differential primer sets comprising at least
 a first primer comprising the first primer sequence, 
 a second primer comprising the second primer sequence, and 
 a third primer, complementary in part to the second primer sequence, but distinguishable from the second primer; 
   (e) amplifying the amplification templates to generate amplicons that correspond to each target sequence and that incorporate either the second primer or the third primer; and   (f) determining the amount of each target sequence based on differential detection of the second and third primers in amplicons corresponding to each target sequence.   
     
     
         126 . The method of  claim 125 , wherein the second and third primers are labeled with different dyes. 
     
     
         127 . The method of  claim 125 , wherein the second and third primers are distinguishable by length. 
     
     
         128 . The method of  claim 125 , wherein the second and third primers are distinguishable by GC content. 
     
     
         129 . The method of  claim 125 , wherein step (e) comprises performing PCR at a lower stringent condition and then at a higher stringent condition. 
     
     
         130 . The method of  claim 125 , wherein the first probe and the second probe hybridize to adjacent domains of a target sequence. 
     
     
         131 . The method of  claim 125 , wherein steps (b) and (c) are an oligonucleotide ligation assay (OLA). 
     
     
         132 . The method of  claim 125 , wherein the first probe and the second probe hybridize to separated domains of a target sequence. 
     
     
         133 . The method of  claim 130 , wherein steps (b) and (c) are an extension-ligation assay. 
     
     
         134 . The method of  claim 125 , wherein steps (b) and (c) are a padlock probe assay. 
     
     
         135 . The method of  claim 125 , wherein the sample is a formalin-fixed, paraffin-embedded (FFPE) sample. 
     
     
         136 . The method of  claim 125 , wherein the sample is a forensic sample. 
     
     
         137 . The method of  claim 125 , wherein the target sequences are expression products. 
     
     
         138 . The method of  claim 137 , wherein the sample comprises cDNAs. 
     
     
         139 . The method of  claim 125 , wherein probe sets for at least 100 different target sequences are provided in step (b). 
     
     
         140 . A multiplex kit, comprising:
 a set of probes for each of at least two target sequences, each probe set comprising
 a first probe having a first target-specific sequence and a first priming sequence, and a second probe having a second target-specific sequence and a second priming sequence; and 
   a differential primer set, comprising at least
 a first primer, complementary in part to the first primer sequence, a second primer, complementary in part to the second primer sequence, and a third primer, complementary in part to the second primer sequence, but distinguishable from the second primer. 
   
     
     
         141 . The kit of  claim 140 , further comprising an enzyme composition. 
     
     
         142 . The kit of  claim 141 , wherein the enzyme composition comprises a ligase. 
     
     
         143 . The kit of  claim 141 , wherein the enzyme composition comprises a polymerase. 
     
     
         144 . The kit of  claim 141 , wherein one of the probes in each probe set comprises an adapter sequence that is specific to the target sequence.